Balakhonova Veronika, Dobisova Tereza, Benedikty Zuzana, Panzarova Klara, Pytela Jaromir, Koci Radka, Spyroglou Ioannis, Kovacova Ingrid, Arnaud Dominique, Skalak Jan, Trtilek Martin, Hejatko Jan
CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czechia.
National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czechia.
Front Plant Sci. 2023 Apr 21;14:1093292. doi: 10.3389/fpls.2023.1093292. eCollection 2023.
Seedling de-etiolation is one of the key stages of the plant life cycle, characterized by a strong rearrangement of the plant development and metabolism. The conversion of dark accumulated protochlorophyllide to chlorophyll in etioplasts of de-etiolating plants is taking place in order of ns to µs after seedlings illumination, leading to detectable increase of chlorophyll levels in order of minutes after de-etiolation initiation. The highly complex chlorophyll biosynthesis integrates number of regulatory events including light and hormonal signaling, thus making de-etiolation an ideal model to study the underlying molecular mechanisms. Here we introduce the iReenCAM, a novel tool designed for non-invasive fluorescence-based quantitation of early stages of chlorophyll biosynthesis during de-etiolation with high spatial and temporal resolution. iReenCAM comprises customized HW configuration and optimized SW packages, allowing synchronized automated measurement and analysis of the acquired fluorescence image data. Using the system and carefully optimized protocol, we show tight correlation between the iReenCAM monitored fluorescence and HPLC measured chlorophyll accumulation during first 4h of seedling de-etiolation in wild type and mutants with disturbed chlorophyll biosynthesis. Using the approach, we demonstrate negative effect of exogenously applied cytokinins and ethylene on chlorophyll biosynthesis during early de-etiolation. Accordingly, we identify type-B response regulators, the cytokinin-responsive transcriptional activators ARR1 and ARR12 as negative regulators of early chlorophyll biosynthesis, while contrasting response was observed in case of EIN2 and EIN3, the components of canonical ethylene signaling cascade. Knowing that, we propose the use of iReenCAM as a new phenotyping tool, suitable for quantitative and robust characterization of the highly dynamic response of seedling de-etiolation.
幼苗脱黄化是植物生命周期的关键阶段之一，其特点是植物发育和代谢发生强烈重排。脱黄化植物的黄化质体中，黑暗中积累的原叶绿素酸酯在幼苗光照后数纳秒到微秒的时间内转化为叶绿素，导致脱黄化开始后几分钟内叶绿素水平可检测到增加。高度复杂的叶绿素生物合成整合了包括光和激素信号在内的许多调控事件，因此使脱黄化成为研究潜在分子机制的理想模型。在此，我们介绍iReenCAM，这是一种新型工具，设计用于以高空间和时间分辨率对脱黄化过程中叶绿素生物合成的早期阶段进行基于荧光的非侵入式定量分析。iReenCAM包括定制的硬件配置和优化的软件包，允许对采集的荧光图像数据进行同步自动测量和分析。使用该系统和精心优化的方案，我们展示了在野生型和叶绿素生物合成受干扰的突变体的幼苗脱黄化的前4小时内，iReenCAM监测的荧光与HPLC测量的叶绿素积累之间的紧密相关性。使用该方法，我们证明了外源施加的细胞分裂素和乙烯在早期脱黄化过程中对叶绿素生物合成的负面影响。相应地，我们确定B型响应调节因子，即细胞分裂素响应转录激活因子ARR1和ARR12是早期叶绿素生物合成的负调节因子，而在典型乙烯信号级联的组分EIN2和EIN3的情况下观察到相反的响应。基于此，我们建议将iReenCAM用作一种新的表型分析工具，适用于对幼苗脱黄化的高度动态响应进行定量和稳健的表征。
